So many of these systems have been developed over the last two or three decades that it is hardly feasible to give a useful summary of the many possibilities. These possibilities are generally known to the people skilled in this art anyway. However, there are a number of genes which are difficult to replicate, transcribe or express for a variety of reasons. A quite obvious reason is for instance that the product produced upon expression is toxic to the cell in which the nucleic acid of interest is expressed. There are however less clear reasons why replication, transcription or expression of a nucleic acid of interest does not lead to useful levels of replication, transcription and/or expression. This invention typically deals with the replication, transcription and/or expression of such nucleic acids. The present invention was made during research involving adeno associated virus (AAV) and is typically useful for replication, transcription and/or expression of nucleic acids in an AAV-based system and typically for replication, transcription and/or expression of AAV-genes, in particular the cap-gene. However, other genes resisting replication, transcription and/or expression in the regular systems or genes or other nucleic acids that may only be produced upon induction will also be suitable for use in the presently invented system. The invention will however ne explained in more detail based on the AAV-system. AAV is a virus that is typically suggested for use in gene therapy, whereby a gene of interest is packaged into an AAV virion, which can infect a cell to be provided with said gene. The present invention arrives at a universal packaging system for AAV derived vectors provided with such a gene therapy related nucleic acid.
AAV is a non-pathogenic human parvovirus (reviewed in.sup.1, 2). The virus replicates as a single strand DNA of approximately 4.6 kb. Both the plus and the minus strand are packaged and infectious. Efficient replication of AAV requires the co-infection of the cell by a helper virus such as Adenovirus or Herpes Simplex Virus. In the absence of a helper virus, no substantial replication of AAV is observed. AAV is therefore also classified as a "Dependovirus", When no helper virus is present, the AAV genome can integrate into the host cell genome. The wild-type virus has a strong preference (70%) for an integration site on the long arm of chromosome 19 (19 q13.3).sup.3-5. Following integration, the expression of the virus genes is not detectable. The integrated provirus replicates as a normal part of the host cell genome upon division of the transduced cell and ends up in both daughter cells. This stage of the virus life cycle is known as the latent stage. This latent stage is stable but can be interrupted by infection of the transduced cell by a helper virus. Following infection of the helpervirus, AAV is excised from the host cell genome and starts to replicate. During the early phase of this lytic cycle the rep-genes are expressed. Approximately 12 to 16 hours later, the capsid proteins VP1, VP2 and VP3 are produced and the replicated virus DNA is packaged into virions (structure of the AAV-genome and its genes is depicted in FIG. 1). The virions accumulate in the nucleus of the cell and are released when the cell lyses as a result of the accumulation of AAV and the helpervirus (reviewed in.sup.1, 2).
The AAV-genome contains two genes named rep and cap (FIG. 1). Three promoters (P5, P19 and P40) drive the synthesis of mRNAs coding for 4 Rep-proteins (Rep78, Rep68, Rep52 and Rep40) and three capsid proteins (VP1, VP2 and VP3). The AAV-genome is flanked on both sides by a 145 bp sequence, called the Inverted Terminal Repeat (ITR), which appears to contain all the cis-acting sequences required for virus integration, replication and encapsidation.sup.6, 7.
The capsid proteins VP1, VP2 and VP3 are produced from a 2.6 kb transcript of the AAV P40 promoter, which is spliced into two 2.3 kb mRNAs by using the same splice donor but two different splice acceptor sites. The splice acceptor sites are located at both sides of the VP1 translation start signal. VP1 is translated from the messenger that uses the splice acceptor directly in front of the VP1 translation initiation codon. VP2 and VP3 are translated from messengers that are spliced to the acceptor 3' of the VP1 ATG. VP2 and VP3 are translated from this messenger by use of an ACG translation start (VP2) or a downstream ATG (VP3). Since all three coding regions are in frame, the capsid proteins share a large domain with an identical amino-acid sequence. VP3 is entirely contained within VP1 and VP2, but the latter two contain additional amino-terminal sequences. Similarly, VP1 contains the entire VP2 protein but carries an additional N-terminal sequence. All three capsid proteins terminate at the same position.sup.8. The AAV capsid is 20 to 24 nm in diameter.sup.9, 10 and contains approximately 5% VP1, 5% VP2 and 90% VP3. This ratio is believed to reflect the relative abundance of the alternatively spliced messengers and the reduced translation initiation efficiency at the ACG initiation codon for VP2.
During a productive infection, the P5-promoter is activated first and directs the production of the large Rep-proteins, Rep78 and Rep68. These proteins are essential for AAV-replication and trans regulation of viral and cellular genes. The large Rep-proteins activate the P19 and the P40 promoter. In a latent infection, however, Rep78 and Rep68 down regulate expression of the P5 promoter and help to maintain the latency of AAV (for a review see.sup.1). The smaller Rep-proteins, Rep52 and Rep40, are encoded by transcripts from the P19 promoter and are important for the formation of infectious virus.sup.11. The P40 promoter is the last promoter to become activated and its activation follows the expression of the late genes of the helper adenovirus. Via alternative splicing, different mRNAs are produced coding for the structural proteins VP1, VP2 en VP3.sup.12.
Adeno-Associated Virus Vector Technology
The first recombinant AAV vectors were made by replacing sequences from the rep or the cap gene by the sequences of interest.sup.13-15. Two methods were used to package the recombinant vector. In one method, the vector genome was packaged by co-transfecting into adenovirus infected cells a plasmid containing the vector together with a plasmid containing the missing AAV-gene. In the second method, a plasmid containing the vector was co-transfected with an AAV-genome that was too large to be packaged by an insertion of lambda phage DNA.sup.13-15. Recombinant virus produced in this way is always contaminated with wild-type AAV (ranging from 10-50% compared to the recombinant titer). This is presumably due to recombination between the two co-transfected plasmids which contain a substantial region of overlap, or by loss of the lambda DNA sequence. The contaminating wild-type AAV causes a further amplification of the rAAV upon infection of a new batch of adenovirus infected cells, leading to higher rAAV-titers but also leading to amplification of the contaminating wild-type AAV.sup.13-15.
To circumvent the production of wild-type AAV, a packaging plasmid was constructed that contains no overlap with the vector plasmid.sup.7. With this packaging plasmid, it is possible to generate rAAV virus stocks that are free of detectable amounts of wild-type AAV, while at the same time it enables the production of 0.1 to 1 rAAV particles per cell.sup.7. This packaging system, or analogous systems derived therefrom, are currently used by most laboratories. Although this is the method of choice at this moment, the method is far from optimal since it cannot easily be scaled up to allow industrial production of rAAV vectors. Plasmid transfections are inherently inefficient and difficult to standardize or to scale up. This is even more true for co-transfections. In addition, whereas the wild-type virus replicates to 10.sup.3 -10.sup.4 particles per cell, the yield of rAAV in a typical rAAV-production is very low with the current methods (01-1 particles per cell).sup.7, 16. This low yield makes purification of the rAAV a difficult task to undertake. The production problems pose a serious technological obstacle for the further development of AAV-vector technology for, for instance, gene therapy purposes. There is clearly a great need for an efficient and simple method for the production of rAAV. A very convenient packaging system would be in vitro packaging of rAAV by purified recombinantly produced AAV-proteins. A practical alternative is the generation of a packaging cell line for rAAV where the packaging cell line supplies in trans the required AAV and helper virus proteins for the production of rAAV. The specific recombinant AAV producing cell lines are then generated by stable transfection of a plasmid containing the recombinant AAV into the packaging cells. The present invention is useful for both the in vitro packaging strategy and the packaging cell line strategy.
Recombinant AAV Packaging Cell Lines
Packaging cell lines are currently the most efficient way in which retrovirus and adenovirus vectors are produced for industrial an/or therapeutic purposes such as gene therapy.sup.17, 18. Virus protein production for the in vitro packaging on an industrial scale is currently employed for Lambda phages, but not for other viruses. The generation of general packaging cell lines for rAAV has been an elusive goal for many years. Recombinant AAV packaging cell lines require that the in trans required AAV-proteins are only functional during the production phase of the rAAV vector. Constitutive function or expression is not desired for at least two reasons: 1) rescue and replication of the vector DNA prior to production would interfere with the growth and the stability of the cell line and 2) specifically the AAV-Rep-proteins are toxic to cells even in the absence of a recombinant AAV vector. The latter is largely due to the well documented, but as yet not explained, anti-proliferative effect of the large Rep proteins.sup.19, 20. Rep78 and Rep68 repress both cellular and viral promoters in transient assays.sup.21, 22. Upon stable transfection, the large Rep proteins inhibit cell proliferation.sup.19, 20. The mechanism is not well understood. It is possible that the observed inhibition of mRNA transcription and translation represses the production of crucial cellular gene-products.sup.23, 25. On the other hand, is it possible that the large Rep-proteins inhibit DNA replication directly.sup.26, 22, 27. Considering the pleiotropic effect of Rep-protein expression on cells, it is possible that both effects play a role in the anti-proliferative effect of the large Rep-proteins.
Cell Lines With Inducible Rep-Gene Expression
Until now, it has not been possible to make stable cell lines expressing the large Rep-proteins constitutively (see above). Following substitution of the P5 promoter with an inducible promoter, such as the methallothionine promoter.sup.20 or the steroid inducible Mouse Mammary Tumor Virus (MMTV) long terminal repeat (LTR) promoter.sup.19, it was possible to isolate out of a large number of clones, one and two clones that inducibly expressed Rep78, respectively. Rep52 was expressed constitutively in two of the three clones, whereas Rep68 and Rep40 which are translated from the spliced mRNAs, were not detectable.sup.19, 20. Although these clones were able to functionally produce Rep78 and Rep52, the levels were too low for the replication and packaging of recombinant AAV.sup.19, 20. Apart from this, the percentage of clones expressing Rep78 was low. Probably, there was a strong selection against a high level of rep expression. In case of the MMTV-promoter driven rep-expression, the replication and production of infectious virus of rep-negative recombinant AAV constructs could be improved by adding a construct constitutively expressing Rep40.sup.28. Still, at least three problems remain.sup.19, 20, 28 : 1) The cell lines do not express capsid proteins. Capsid proteins need to be supplied through transfection of a capsid gene construct. 2) Significant replication of rAAV-constructs requires transfection of the glucocorticoid receptor (in case of the MMTV-promoter). 3) The yield of rAAV is not improved over the transient packaging systems and thus is not sufficient for industrial use in the sense of production of gene delivery vehicles.
Recently, we were able to generate cell lines with inducible and high level rep-gene expression using an improved inducible promoter system. However, also for these cell lines the capsid genes need to be added externally during virus production.
Recently, Clark et al. reported the generation of a full complementing cell line.sup.29. Although they do not know how to reconcile their results with the results of most other laboratories, they succeeded in generating cell lines inducibly expressing rep and cap from constructs that were stably integrated into the host cell genome. Unfortunately, this result was only obtained when the rep-gene, the cap-gene, a dominant selectable marker gene and the rAAV-vector sequences were present on the same plasmid, thus resulting in dedicated packaging cell lines. These packaging cells can, therefore, only be used for the production of the specific rAAV introduced together with the rep and cap genes and not be used to produce a different rAAV vector. It was also attempted to generate a general packaging cell line.sup.29. A cell line was obtained that inducibly replicated introduced rAAV and expressed the cap-gene. However, the levels of rep-expression where significantly lower than in the dedicated cell lines and although cap-RNA was produced, the levels were insufficient to make this cell line suitable for packaging of recombinant AAV. Since the rep and the cap-genes are physically linked to each other in this approach, it is not likely that the levels of rep and cap can easily be enhanced. For instance, there is the risk that rep-gene expression in the uninduced state is elevated to a level toxic for the packaging cells. This cell line was intended for and is useful for determining the infectious titer of rAAV preparations and testing of new rAAV vectors in a transient assay.sup.29, 30.